murine fgf21  (Proteintech)

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: ( A ) Schematic illustration of housing conditions and experimental timeline. ( B ) Concentrations of FGF21 in plasma pretransduction (Pre) and at euthanasia (Euth). ( C ) Concentrations of sTGFBR2 in plasma 3, 5, 7, and 9 weeks after transduction. Body weight over time in 60% HFD-fed female mice housed at ( D ) 22°C, ( E ) 26°C, or ( F ) 30°C ( n = 6–7). Insulin tolerance tests (0.75 U/kg) in mice housed at ( G ) 22°C, ( H ) 26°C, or ( I ) 30°C ( n = 6–7), data presented as mean ± SEM. #Mice in G were given intraperitoneal glucose because their blood glucose concentrations dropped below 30 mg/dL. ( J ) Plasma TAG concentrations 8 weeks posttreatment in mice housed at 22°C, 26°C, or 30°C ( n = 6–7). * P < 0.05. Statistical analyses were performed using 2-way ANOVA, followed by Bonferroni’s post hoc test. TAG, triacylglycerol.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques: Clinical Proteomics, Transduction

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: Female mice were euthanized 10 weeks after transduction with FGF21/sTGFBR2 ( n = 5–7). ( A ) Body weight of mice transduced with FGF21/sTGFBR2 at euthanasia. ( B ) Posterior subcutaneous white adipose tissue (psWAT) and ( C ) brown adipose tissue (BAT) weights. ( D ) Representative histological images of mouse BAT stained with H&E. Scale bar = 50 μm. Fed ( E ) blood glucose concentrations, ( F ) plasma TAG, and ( G ) insulin concentrations prior to euthanasia. Tibial ( H ) cortical thickness (Ct.Th), ( I ) cortical area (Ct.Ar), and ( J ) cortical bone volume fraction (Ct.BV/TV) analyzed via nanoCT. * P < 0.05. Statistical analyses were performed using 2-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques: Transduction, Staining, Clinical Proteomics

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: ( A ) Liver weights in vehicle- and FGF21/sTGFBR2-treated mice at all housing temperatures 10 weeks after transduction ( n = 6–7). ( B ) Liver TAG concentration normalized to protein amount ( n = 6–7). ( C ) Representative histological images of mouse livers stained with H&E. Scale bar = 50 μm. ( D ) Representative images of mouse livers stained with Picrosirius red to identify collagen. Scale bar = 50 μm. * P < 0.05. Statistical analyses were performed using 2-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques: Transduction, Concentration Assay, Staining

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: ( A ) Schematic illustration of housing conditions and experimental timeline; Lmna fl/fl = control mice and Lmna ADKO = lipodystrophic mice. Plasma ( B ) FGF21 and ( C ) sTGFBR2 concentrations in mice 12 weeks after transduction ( n = 4–7). ( D ) Body weight in vehicle and FGF21/sTGFBR2 mice 4 weeks after transduction ( n = 5–7). ( E ) Fat and ( F ) lean mass measured by EchoMRI 4 weeks after transduction ( n = 4–7). ( G ) Plasma TAG and ( H ) blood glucose concentrations 4 weeks posttreatment ( n = 5–7). ( I ) Insulin tolerance tests (0.75 U/kg; n = 5–7), data presented as mean ± SEM. * P < 0.05. Statistical analyses were performed using 2-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques: Control, Clinical Proteomics, Transduction

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: Female mice were euthanized 12 weeks after FGF21/sTGFBR2 transduction ( n = 4–7). ( A ) Body, ( B ) psWAT, ( C ) parametrial white adipose tissue (pmWAT), ( D ) BAT, ( E ) spleen, and ( F ) heart weights in mice at euthanasia. Fed ( G ) blood glucose, ( H ) plasma TAG, and ( I ) plasma insulin concentrations prior to euthanasia. ( J ) Representative tibial osmium-nanoCT images. ( K ) rBMAT normalized to marrow volume. ( L ) cBMAT normalized to marrow volume. * P < 0.05. Statistical analyses were performed using 2-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques: Transduction, Clinical Proteomics

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: Female mice were euthanized 12 weeks after FGF21/sTGFBR2 transduction ( n = 4–7). ( A ) Liver weights at euthanasia. ( B ) Liver TAG normalized to protein content. ( C ) TAG amount normalized to total liver weight. ( D ) Representative histological images of mouse livers stained with H&E. Scale bar = 50 μm. ( E ) Representative images of mouse livers stained with Picrosirius red for collagen. Scale bar = 50 μm. ( F ) Machine learning quantification of area stained by Picrosirius red in the liver. Statistical analyses were performed using 2-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques: Transduction, Staining

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: ( A ) Schematic illustration of housing conditions and experimental timeline. ( B ) Plasma FGF21 concentrations 8 weeks post-transduction ( n = 3–16). ( C ) Body weight, ( D ) fat mass, and ( E ) lean mass measured by EchoMRI 7 weeks posttransduction ( n = 3–13). ( F ) Glucose tolerance test (1 mg/kg; n = 3–13), data presented as mean ± SEM. ( G ) Insulin tolerance test (0.75 U/kg; n = 3–13), data presented as mean ± SEM. #Mice in G received intraperitoneal glucose because their blood glucose dropped below 30 mg/dL. Indirect calorimetry was measured for 3 days using the Promethion system. ( H ) Energy expenditure (EE) averaged during dark or light cycles, ( I ) fat oxidation averaged during the first 4 hours of dark or light cycles, and ( J ) distance traveled averaged during dark or light cycles. * P < 0.05. Statistical analyses were performed using 2-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques: Clinical Proteomics, Transduction

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: Female mice were euthanized 8 weeks after FGF21 administration ( n = 3–15). ( A ) Body, ( B ) psWAT, ( C ) pmWAT ( D ) BAT, and ( E ) heart weights at euthanasia. Fed ( F ) blood glucose, ( G ) plasma TAG, ( H ) plasma insulin, and ( I ) plasma leptin concentrations prior to euthanasia. * P < 0.05. Statistical analyses were performed using 2-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques: Clinical Proteomics

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: Female mice were euthanized 8 weeks after FGF21 administration ( n = 3–15). ( A ) Liver weights at euthanasia. ( B ) Liver TAG normalized to protein content. ( C ) Total liver TAG normalized to liver weights. ( D ) Representative histological images of H&E-stained mouse livers. Scale bar = 50 μm. ( E ) Representative images of mouse livers stained with Picrosirius red for collagen. Scale bar = 50 μm. ( F ) Machine learning quantification of area stained by Picrosirius red in the liver. * P < 0.05. Statistical analyses were performed using 2-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques: Staining

Journal: JCI Insight

Article Title: Effects of FGF21, soluble TGFBR2, and environmental temperature on metabolic dysfunction in lipodystrophic mice

doi: 10.1172/jci.insight.194882

Figure Lengend Snippet: ( A ) Summary of key GSEA pathways in hepatic mRNA of CTRL versus ADKO mice 8 weeks after treatment with FGF21 or vehicle. ( B ) GSEA pathways changed in CTRL mice dependent on treatment, padj < 0.05. ( C ) GSEA pathways changed in ADKO mice dependent on treatment, padj < 0.05. ( D ) GSEA pathways changed in vehicle-treated mice dependent on genotype, padj < 0.05. ( E ) GSEA pathways changed in FGF21-treated mice dependent on genotype, padj < 0.05.

Article Snippet: ELISA was used to measure circulating concentrations of leptin (900-K76K, Invitrogen), insulin (90080, Crystal Chem), murine FGF21 (MF2100, R&D Systems), and murine sTGFBR2 (ab277719, Abcam).

Techniques:

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a An overview of a general workflow for the study. b Comparison of pre- and post-transplantation Fgf21 mRNA expression in the liver graft based on the GSE15480 (n = 12 pairs, including pre- and post-LT donor livers) and GSE151648 (n = 40 pairs, including pre- and post-LT donor livers) derived from graft biopsies in liver transplantation. c Comparison of pre- and post-reperfusion FGF21 levels in the serum (Cohort 1, n = 88 pairs, including pre- and 2 h post-LT recipient serum). d The peripheral FGF21 level 2 h after reperfusion linearly correlated with maximal ALT within 7 days after transplantation. e The 88 patients were divided into the FGF21-elevated group (n = 44) and non-elevated group (n = 44) according to the median value of ratio change (post-reperfusion/pre-transplant). The elevated group had improved graft survival. f , g Pre-transplant FGF21 expression in biopsies (Cohort 2, n = 115) and its correlation with graft survival (after excluding liver grafts with CIT > 15 h, n = 107). h Information on graft steatotic change was missing for 19 cases in this cohort. We confirmed that 33 patients received steatotic liver grafts. Low FGF21 in the graft was associated with elevated ALT and AST after transplantation. However, there was no significant difference in the non-steatotic subgroup (n = 55). I/R, ischemia/reperfusion; KO knockout, ALT alanine aminotransferase, AST aspartate aminotransferase, CIT cold ischemia time. p values are shown on the graphs. Source data are provided as a file. Figure 1a created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Comparison, Transplantation Assay, Expressing, Derivative Assay, Knock-Out

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a The time-dependent western blot analysis (left) and quantification (right) of FGF21 protein expression in the mouse liver undergoing ischemia for 1.5 h followed by the indicated duration of reperfusion, three independent biological mice samples. b Schematic for the establishment of the mouse I/R injury model. c Serum ALT and AST levels of wild-type and Fgf21 KO mice in the sham and I/R groups 6 h after reperfusion (n = 5, per group). d Representative H&E staining of liver sections from wild-type and Fgf21 KO mice in the sham and I/R groups. e Liver damage was evaluated using Suzuki’s histological score (n = 5, per group). f Representative TUNEL immunofluorescent staining in liver lobes of wild-type and Fgf21 KO mice in the sham and I/R groups. g Quantification analysis of the TUNEL-positive cells/high-power field (n = 5, per group). h Volcano plot of the differentially expressed genes (DEGs) between wild-type and Fgf21 KO livers after I/R treatment (n = 4) using the absolute value of log2 (fold change) >1.5 and p < 0.05 as the thresholds. i KEGG pathway enrichment analysis of the identified DEGs. j Heatmap of major metabolites catalyzed by lipoxygenases in wild-type and Fgf21 KO livers after I/R treatment (n = 4). The 15-HETE content was elevated in Fgf21 KO livers. k Co-expression network and KEGG pathway analysis of the hub metabolites. l The Pearson correlation coefficients between ALOX15/15-HETE (RNA-seq/metabolomic) and the co-expression modules identified by WGCNA. KEGG analysis of the module with the highest correlation coefficients ( p < 0.05 by Fisher’s exact test, two-sided). WT, wild-type, KO knockout, I/R ischemia/reperfusion, ALT alanine aminotransferase, AST aspartate aminotransferase. a , c , e , g , j Two-tailed t test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 2b created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Western Blot, Expressing, Staining, TUNEL Assay, RNA Sequencing, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a Wild-type and Fgf21 KO donor livers in the mouse orthotopic liver transplantation model were harvested, stored for 30 min at 4 °C, and transplanted into wild-type recipient mice. b Serum ALT and AST levels for each group after transplantation (n = 4, per group). c The 15-HETE level in the livers of each group 6 h after reperfusion (n = 4, per group). d The FGF21 level in the serum of each group 6 h after reperfusion (n = 4, per group). e Representative TUNEL staining of liver sections from each group (n = 4, per group). f Heatmap showing the differential expression of 41 immune markers in 34 cell clusters. g Two-dimensional t-SNE illustration of the CyTOF data of livers isolated from WT >> WT and KO >> WT (n = 4/group), the merged t-SNE graph was used for contrast. h – j The frequencies of the identified clusters in granulocytes, Mono/Mac cells and NK cells and the comparisons between the groups (n = 4, per group), boxplot shows the median (center line), 25th, and 75th percentile (lower and upper boundary), and the whiskers are essentially range bars that extend to max and min values. k Multiplex immunohistochemistry staining of NK1.1 (green), CD62L (red) and DAPI (blue) in Fgf21 KO-transplanted livers, three independent biological mice samples. Scale bar, 50 μm. OLT orthotopic liver transplantation, WT wild-type, KO knockout, ALT, alanine aminotransferase, AST aspartate aminotransferase, TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling, Mono/Mac monocyte/macrophage, NK natural killer. Two-tailed t-test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 4a created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Transplantation Assay, TUNEL Assay, Staining, Quantitative Proteomics, Isolation, Multiplex Assay, Immunohistochemistry, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a Experimental schema for establishing the I/R injury mouse model. The mice were intravenously administered rmFGF21 (0.5 mg/kg). b Serum ALT and AST levels 6 h after reperfusion (n = 5, per group). c Comparison of serum FGF21 levels 6 h after reperfusion (n = 5, per group). d H&E staining of liver sections 6 h after reperfusion (n = 5, per group). e Western blot analysis (left) and quantification (right) of FGF21and BAX in AML12 from each group. f Western blot analysis (left) and quantification (right) of ALOX15, BAX and p-ERK1/2 in AML12 from each group. g Western blot analysis (up) and quantification (down) of ALOX15, BAX and p-FGFR4 in AML12 from each group. e – g three independent biological mice samples. I/R ischemia/reperfusion, ALT alanine aminotransferase, AST aspartate aminotransferase, H/R hypoxia/ reoxygenation. Two-tailed t-test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 5a created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Comparison, Staining, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a IHC staining for FGF21 in pre-transplant biopsies from the steatotic liver grafts in Cohort 3 (n = 78). b Comparison of the degree of hepatic steatosis between the low FGF21 group and the high FGF21 group. c Comparison of maximal serum ALT/AST within 7 days after transplantation between the low FGF21 group and the high FGF21 group. d Comparison of FGF21 staining (mean integrated optical density, IOD) between patients who did and did not develop EAD. e Schematic for the establishment of the I/R injury model using mice fed a high-fat diet (HFD) or normal chow diet. After 8 weeks of feeding, mice were subjected to I/R treatment. f Serum ALT and AST 6 hours after reperfusion (n = 5, per group). g 15-HETE content in the livers from each group 6 h after reperfusion (n = 5, per group). h H&E staining, ROS staining and TUNEL staining of liver sections from each group 6 h after reperfusion (n = 5, per group). i Quantification assessment of infiltrating CD11b + and MPO + cells in the livers from each group (n = 5, per group). I/R ischemia/reperfusion, WT wild-type, KO knockout, HFD high-fat diet, ALT alanine aminotransferase, AST aspartate aminotransferase, HE hematoxylin and eosin, ROS reactive oxygen species, DHE dihydroethidium, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, MPO cytosolic myeloperoxidase. Two-tailed t-test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, ns, not significant, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 6e created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Immunohistochemistry, Comparison, Transplantation Assay, Staining, TUNEL Assay, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a The steatotic mouse models underwent intravenous injection of rmFGF21 (0.5 mg/kg) or tail vein injection of AAV8-hAAT- Fgf21 before I/R injury. b 15-HETE content in the livers from each group 6 h after reperfusion (n = 4, per group). c Serum ALT and AST 6 h after reperfusion (n = 5, per group). d H&E staining, ROS staining and TUNEL staining and quantification results of liver sections from each group 6 h after reperfusion (n = 5, per group). e Quantification assessment of infiltrating CD11b + and MPO + cells in the livers of each group (n = 5, per group). f The general schematic diagram. LT, liver transplantation; AA, arachidonic acid. I/R, ischemia/reperfusion; HFD, high-fat diet; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HE, hematoxylin and eosin; ROS, reactive oxygen species; DHE, dihydroethidium; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; MPO, cytosolic myeloperoxidase. Two-tailed t-test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 8a, f created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Injection, Staining, TUNEL Assay, Transplantation Assay, Two Tailed Test

Journal: International journal of molecular sciences

Article Title: Recombinant FGF21 Attenuates Polychlorinated Biphenyl-Induced NAFLD/NASH by Modulating Hepatic Lipocalin-2 Expression.

doi: 10.3390/ijms23168899

Figure Lengend Snippet: Figure 1. PCB exposure induces NAFLD/NASH in mice fed either a SD and a HFD. Eight-week-old male C57BL/6 mice were fed either a SD or HFD for four weeks and then treated with vehicle (corn oil), Aroclor1260 (20 mg/kg) or PCB126 (5 mg/kg) by intraperitoneal injection for a total of four injections (two, three, four, and five weeks) during the six-week study duration. (A) Experimental design. (B) Representative images of H&E staining of liver tissue. Scale bar: 50 µm. (C) Quantification of the lipid droplet area in H&E-stained liver tissue. The data are expressed as a percentage of the area of lipid droplets in the field. (D) Hepatic TG. (E) Hepatic FFA. (F) Plasma FGF21. (G) ORO staining showing that both Aroclor1260 (20 µM) and PCB126 (10 µM) accelerated hepatic lipid accumulation in O/P-treated human ARE primary hepatocytes. Scale bar: 50 µm. (H) Representative images of H&E staining of adipose tissue. The crown-like structure is illustrated by the asterisk. Scale bar: 50 µm. (I) Average adipocyte size of eWAT was measured in H&E images using ImageJ 1.53 s. (J) Quantitative analysis of CLS formation in adipose tissue. All values represent the mean ± SD, n = 7–10 mice per group. * p < 0.05 and ** p < 0.01.

Article Snippet: Recombinant human FGF21 (rhFGF21) was purchased from Peprotech (Rocky Hill, NJ, USA), and recombinant murine FGF21 (rmFGF21) was purchased from Cusabio (Houston, TX, USA).

Techniques: Injection, Staining, Clinical Proteomics

Journal: International journal of molecular sciences

Article Title: Recombinant FGF21 Attenuates Polychlorinated Biphenyl-Induced NAFLD/NASH by Modulating Hepatic Lipocalin-2 Expression.

doi: 10.3390/ijms23168899

Figure Lengend Snippet: Figure 4. Recombinant FGF21 improves hepatic steatosis in PCB-induced NAFLD/NASH models. Aroclor1260- or PCB126-injected mice fed either a SD or HFD were intraperitoneally administered vehicle or rmFGF21 (1 mg/kg/day) once daily for 10 days. (A,B) Representative photograph images. (C,D) Liver weight. (E) H,E staining of liver tissue. Scale bar: 50 µm. (F) ORO staining and quantification showing that rhFGF21 (50 ng/mL, 24 h) reduced the hepatic lipid accumulation induced by both 20 µM Aroclor1260 and 10 µM PCB126 in O/P-treated human ARE primary hepatocytes. Scale bar: 50 µm. All values represent the mean ± SD, n = 4. * p < 0.05, ** p < 0.01 compared with the experimental control.

Article Snippet: Recombinant human FGF21 (rhFGF21) was purchased from Peprotech (Rocky Hill, NJ, USA), and recombinant murine FGF21 (rmFGF21) was purchased from Cusabio (Houston, TX, USA).

Techniques: Recombinant, Injection, Staining, Control

Journal: International journal of molecular sciences

Article Title: Recombinant FGF21 Attenuates Polychlorinated Biphenyl-Induced NAFLD/NASH by Modulating Hepatic Lipocalin-2 Expression.

doi: 10.3390/ijms23168899

Figure Lengend Snippet: Figure 5. Recombinant FGF21 attenuates hepatic iron overload in PCB-induced NAFLD/NASH models. Aroclor1260 (20 mg/kg)- or PCB126 (5 mg/kg)-injected mice fed either a SD or HFD were intraperitoneally administered vehicle or rmFGF21 (1 mg/kg/day) once daily for 10 days. (A) Prussian blue staining showing marked iron accumulation in liver samples. Scale bar: 50 µm. (B) Hepatic iron levels in PCB-induced NAFLD/NASH mice that were intraperitoneally administered vehicle or rmFGF21 (left panel, ferrous ion level; right panel, ferrous and ferric iron total levels). All values represent the mean ± SD, n = 7–10 mice per group. * p < 0.05 and ** p < 0.01. (C) Detection of intracellular ferrous ions (Fe2+) using the fluorescent probe FerroFarRed in O/P- and FAC-cotreated HepG2 cells. Scale bar: 200 µm.

Article Snippet: Recombinant human FGF21 (rhFGF21) was purchased from Peprotech (Rocky Hill, NJ, USA), and recombinant murine FGF21 (rmFGF21) was purchased from Cusabio (Houston, TX, USA).

Techniques: Recombinant, Injection, Staining

Journal: International journal of molecular sciences

Article Title: Recombinant FGF21 Attenuates Polychlorinated Biphenyl-Induced NAFLD/NASH by Modulating Hepatic Lipocalin-2 Expression.

doi: 10.3390/ijms23168899

Figure Lengend Snippet: Figure 7. Recombinant FGF21 reduces PCB-induced overexpression of hepatic LCN2. Ten days after the rmFGF21 administration (1 mg/kg/day) in Aroclor1260- or PCB126-induced NAFLD/NASH mice, (A) real-time PCR and (B) western blot analysis were performed to detect mouse LCN2 in liver tissues from mice fed either a SD or HFD. The values represent the mean ± SD, n = six to eight mice per group. * p < 0.05 and ** p < 0.01. (C) Western blot analysis was performed to assess human LCN2 in 20 µM Aroclor1260- or 10 µM PCB126-treated HepG2 cells cotreated with either vehicle or rhFGF21 (50 ng/mL, 24 h).

Article Snippet: Recombinant human FGF21 (rhFGF21) was purchased from Peprotech (Rocky Hill, NJ, USA), and recombinant murine FGF21 (rmFGF21) was purchased from Cusabio (Houston, TX, USA).

Techniques: Recombinant, Over Expression, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Browning marker gene quantification by qPCR in epididymal white adipose tissue (WAT) from WT mice fasted 0–48 h. n = 6 mice for each group. Circles, 0 h fast; squares, 12 h fast; upward triangle, 24 h; downward triangle, 48 h. B NAMPT, SIRT1, and FGF21 gene expression in livers from WT mice fasted 0–48 h. n = 6 mice for each group. Symbols: Circles, fasting 0 h. Squares, 12 h. Upward triangles, 24 h, downward triangle, 48 h. C Representative (from n = 4 per group) NAMPT immunoblot in livers from WT mice fasted 0–48 h. D qPCR quantification of NAMPT, SIRT1, FGF21 gene expression in primary hepatocytes treated with the fasting-mimetic glucose transporter inhibitors, trehalose and lactotrehalose. n = 4 per group. Circles, cultures treated with regular growth media, Red triangles, trehalose-treated, Blue squares, LactoTrehalose-treated. E qPCR in liver from WT mice treated with 5-day 3% trehalose ad libitum in drinking water. n = 8 vehicle- (black circles) and 18 trehalose-treated (red squares). F Fasting-inducible NAMPT expression by qPCR in isolated primary hepatocytes treated with or without 10 mM fructose in the presence or absence of carbohydrate transporter inhibitors trehalose or lactotrehalose. n = 3 starved, 4 fructose-treated, 4 trehalose- and fructose-treated, 4 lactotrehalose- and fructose-treated. Circles, cultures treated with regular growth media; squares, fructose-treated; downward triangles, cultures treated with trehalose and fructose; open circles, LactoTrehalose and fructose-treated cultures. Error bars in ( A – B ), ( D – F ) represent standard error of the mean (SEM). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Statistical tests: ( A – F ), two-tailed T -test, Bonferroni–Dunn post hoc.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Marker, Expressing, Western Blot, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Heat-Zeitgeber Time tracing in WT and NAMPT LKO littermate mice during light and dark cycles during fasting. Mean heat production in WT and NAMPT LKO mice during light and dark cycle fed and early (Fasting I, 1- 24 h) and prolonged (Fasting II, 24–48 h) fasting periods is graphed below. n = 3 WT, 4 NAMPT LKO mice. B RER-Zeitgeber time tracing in WT and NAMPT LKO littermate mice during light and dark cycles during ad-lib fed (first 24 h) and fasting periods. Mean RER in WT and NAMPT LKO mice during light and dark cycle fed and fasting periods is graphed below. n = 3 WT, 4 NAMPT LKO mice. Error bars in ( A – B ) (bottom panels) represent SEM. Circles, WT mice. Squares, NAMPT LKO mice for ( A and B ). C Liver triglycerides (TG), total cholesterol and non-esterified fatty acid (FFA) quantification following 48 h fasting in WT and NAMPT LKO mice. n = 4 WT Fed, 6 WT Fasting; 4 NAMPT LKO Fasting. D qPCR quantification of liver FGF21, NAMPT in fed and 48 h-fasting WT and NAMPT LKO mice. n = 4 WT Fed, 6 WT Fasting; 4 NAMPT LKO Fasting. Error bars in ( C and D ). represent SEM. Black circles, Fed WT, Red circles, fasting WT, Blue circles, Fasting NAMPT LKO mice. E Venn diagram representing significantly altered genes ( P < 0.05) in random-fed vs. 12 h fasting WT and NAMPT LKO mice, quantified by unbiased transcriptomics. n = 2 WT Fed, 4 WT Fasting; 5 NAMPT LKO Fasting. F Bar plot demonstrating −log(FC) for significantly down-regulated gene ontology (GO) pathways revealed when comparing WT vs. NAMPT LKO livers ( P < 0.05). n = 4 WT Fed, 6 WT Fasting; 4 NAMPT LKO Fasting. Statistical tests: ( A ) (upper panel), ANCOVA. A (lower panels), ( B – D ), two-tailed T -test, Bonferroni–Dunn post hoc correction. E , F EdgeR Exact, Benjamini–Hochberg post hoc correction.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Experimental design for db/db AdNAMPT analyses. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. B Body weight over time in db/db mice expressing GFP or NAMPT. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. C Body composition by echoMRI analysis in mice expressing GFP or NAMPT. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. D Serum triglycerides (TG), cholesterol and low-density lipoprotein cholesterol (LDL-C) in db/db mice treated with adenovirus to overexpress GFP or NAMPT. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. E RNAseq quantification heatmap, arranged by calculated hierarchical clustering, demonstrating genes P < 0.05 in db/db AdNAMPT vs. AdGFP mice. n = 4 db/db AdGFP; 4 db/db AdNAMPT mice. Color scale represents Log(FC) ( F ). Most significantly altered KEGG pathways demonstrating log(FC) between db/db AdNAMPT and db/db AdGFP livers. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Unadjusted P < 0.05 for all pathways shown. G qPCR confirmation of RNAseq data suggesting upregulated liver FGF21 for n = 10 db/db AdGFP; 9 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. H and I FGF21 quantification by immunoblot analysis and serum ELISA in db/db AdGFP and db/db AdNAMPT mice. n = 3 db/db AdGFP; 3 db/db AdNAMPT mice for immunoblot data. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice for ELISA data. * P < 0.05, ** P < 0.01 vs. AdGFP. J Glucose tolerance testing and area under the GTT curve in db/db mice treated with adenovirus encoding GFP or NAMPT. n = 9 db/db AdGFP; 9 db/db AdNAMPT biologically independent mice. K Insulin tolerance testing and % area over the ITT curve in db/db mice treated with adenovirus encoding GFP or NAMPT. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. In ( G – K ), Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars in ( G , H ) (left), ( I – K ) represent SEM. Statistical tests: ( B , J , K ), repeated measures ANOVA. C , D , G , H , I , J (right panel), ( K ) (right panel) two-tailed T -test. F EdgeR Exact, Benjamini–Hochberg post hoc correction.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Body weights over time in WT and SIRT1 LKO mice overexpressing empty vector or NAMPT on 12wk Western diet. n = 3 SIRT1 fl/fl Chow; 9 SIRT1 fl/fl EV WD; 6 SIRT1 fl/fl AAV8-NAMPT WD; 5 SIRT1 LKO EV WD. B – D 4 h fasting glucose, insulin, and HOMA-IR (homeostatic model of insulin resistance − fasting glucose (mg/dL) × fasting insulin (ng/mL)/405) in WT and SIRT1 LKO mice overexpressing vector or NAMPT after 12wk chow or Western diet feeding. n = 4 SIRT1 fl/fl Chow; 11 SIRT1 fl/fl EV WD; 4 SIRT1 LKO EV WD; 6 SIRT1 LKO AAV8-NAMPT WD. E FGF21 mRNA and serum peptide, as quantified by qRT-PCR in livers and by serum ELISA in WT and SIRT1 LKO mice overexpressing empty vector or NAMPT after Western diet feeding. Interaction, P < 0.01 and <0.05 for FGF21 mRNA and peptide, respectively. n = 4 SIRT1 fl/fl Chow; 11 SIRT1 fl/fl EV WD; 6 SIRT1 fl/fl AAV8-NAMPT WD; 4 SIRT1 LKO EV WD; 6 SIRT1 LKO AAV8-NAMPT WD ( F ). Indirect calorimetry in WT and SIRT1 LKO mice overexpressing vector or NAMPT after 12wk chow or Western diet feeding. n = 3 SIRT1 fl/fl Chow; 6 SIRT1 fl/fl EV WD; 6 SIRT1 fl/fl AAV8-NAMPT WD; 4 SIRT1 LKO EV WD; 5 SIRT1 LKO AAV8-NAMPT WD. Interactions for light and dark cycle heat: P = ns, and P < 0.01 respectively. *, **, P < 0.05, <0.01 vs. bracketed control by Tukey’s multiple comparisons testing. Error bars in ( A – F ) represent SEM. Circles, SIRT1 fl/fl Chow; Squares, SIRT1 fl/fl EV WD; Upward Triangles SIRT1 fl/fl AAV8-NAMPT WD; Downward Triangles, SIRT1 LKO EV WD; Diamonds SIRT1 LKO AAV8-NAMPT WD. Statistical tests: ( A ), repeated measures ANOVA. B – D one-way ANOVA with Dunnett’s post hoc testing. E , F Two-way ANOVA, Tukey’s post hoc correction.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Plasmid Preparation, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Volcano plots showing differentially expressed genes from (threshold log(FC) = 2, FDR < 0.05) livers from WD-fed SIRT1 fll/fl NAMPT-overexpressing mice and WD-fed SIRT1 fll/fl vector controls (at left). Right, volcano plot showing differentially-expressed genes in livers from WD-fed NAMPT-overexpressing SIRT1 LKO mice vs. NAMPT-overexpressing SIRT1 fll/fl mice. n = 3 WD-fed SIRT1 fll/fl EV; 3 WD-fed SIRT1 fll/fl NAMPT; 3 WD-fed SIRT1 LKO AAV8-NAMPT. B Gene count of differentially expressed genes in livers from WD-fed SIRT1 fll/fl AAV-NAMPT vs. WD-fed SIRT1 fll/fl vector control mice (Red); differentially expressed genes in WD-fed SIRT1 LKO mice and WD-fed SIRT1 fll/fl mice overexpressing NAMPT (Blue). Common gene count is shown in their intersection (purple). n = 3 WD-fed SIRT1 fll/fl EV; 3 WD-fed SIRT1 fll/fl NAMPT; 3 WD-fed SIRT1 LKO AAV8-NAMPT. C Left, Unsupervised clustering of differentially expressed genes (Western diet-fed AAV8-NAMPT vs. Western diet-fed AAV8-NAMPT × SIRT1 LKO ). Right, pathway-based gene expression heatmap of the PI3K/AKT signaling pathway, a significantly upregulated GO pathway and CompBio NAMPT-SIRT1-dependent theme. Log 2 (FC) values are Western diet-fed AAV8-NAMPT vs. Western diet-fed AAV8-EV. n = 3 WD-fed SIRT1 fll/fl EV; 3 WD-fed SIRT1 fll/fl NAMPT; 3 WD-fed SIRT1 LKO AAV8-NAMPT. Heatmap color scales represent Log(FC). D Working model of hepatocyte NAMPT signaling and function. NAMPT nicotinamide phosphoribosyltransferase, SIRT1 sirtuin 1, FGF21 fibroblast growth factor 21, GLUT glucose transporter. Statistical tests: ( A , B , D ) EdgeR Exact, Benjamini–Hochberg post hoc correction.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Plasmid Preparation, Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 1. Effects of 3-O-acetyloleanolic acid isolated from Forsythiae Fructus on the secretion of FGF21 in C2C12 myotubes. (A) Outline of the extraction and isolation of 3-O-acetyloleanolic acid from Forsythiae Fructus. (B) Structure of 3-O-acetyloleanolic acid. (C) The secretion of FGF21 was increased by 3-O-acetyloleanolic acid isolated from Forsythiae Fructus in C2C12 myotubes. Each value is the mean ± SEM of four separate experiments. The double asterisk indicates P < 0.01.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Isolation, Extraction

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 2. 3-O-Acetyloleanolic acid up- regulates FGF21 expression in C2C12 myotubes. (A) The secretion of FGF21 was increased by 3-O-acetyloleanolic acid in a concentration-dependent manner in C2C12 myotubes. Cells were treated with the indicated concentration of 3-O-acetyloleanolic acid for 20 h, then the media were collected and pro cessed for ELISA. Each value represents the mean ± SEM of five separate ex periments. **P < 0.01, comparison with vehicle control. (B) FGF21 mRNA expression was up-regulated by 3-O- acetyloleanolic acid in C2C12 myo tubes. Cells were treated with 30 µM 3- O-acetyloleanolic acid for 2 and 5 h, harvested, and then processed for real- time qRT-PCR. Each value represents the mean ± SEM of five separate ex periments. *P < 0.01; **P < 0.01, com parison with vehicle control.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 3. 3-O-Acetyloleanolic acid stimulates FGF21 expression in mice. (A) Effect of the intraperitoneal administration of 3-O-acetyloleanolic acid at 30 mg/kg on FGF21 mRNA levels in soleus muscle. Total RNA was isolated from the soleus muscle of saline-treated and 3-O-acetyloleanolic acid-treated mice. FGF21 mRNA was determined by qRT-PCR using specific primers. Relative mRNA levels were normalized to 18S. Each value is the mean ± SEM of five mice. The single asterisk indicates P < 0.05. (B) 3-O-acetyloleanolic acid at 30 mg/kg increased circulating FGF21. The plasma FGF21 concentration was determined by FGF21-specific ELISA after the intraperitoneal administration of 3-O-acetyloleanolic acid at 30 mg/kg. Each value represents the mean ± SEM of five mice. The double asterisk in dicates P < 0.01.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Expressing, Isolation, Saline, Quantitative RT-PCR, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 5. Role of p38 MAPK in the up-regulation of FGF21 secretion caused by 3-O-acetyloleanolic acid in C2C12 cells. (A) Cells were treated with 30 µM 3-O-ace tyloleanolic acid for 10 min, harvested, and then processed for western blotting analysis of the phosphorylated and total p38 levels in whole cell lysate. Western blots were quantified using ImageJ software (National Institutes of Health). Each value represents the mean ± SEM of four to five separate experiments. **P < 0.01, comparison with vehicle control. (B) Relative luciferase activity of the FGF21 promoter construct (mFgf21pro1-luc) in C2C12 cells following treatment with 3-O- acetyloleanolic acid and co-transfection of the TGR5-expression plasmid. The total amount of DNA transfected was standardized with an empty vector. After transfection, the cells were preincubated with the indicated concentrations of SB203580 for 20 min and then stimulated with 30 μM 3-O-acetyloleanolic acid for 8 h. Luciferase activity was normalized by β-gal and expressed relative to that in vehicle-treated cells. Each value is the mean ± SEM of five independent experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of SB203580 concentrations. (C) Cells were incubated for 20 h with 3-O- acetyloleanolic acid and SB203580, and FGF21 production was determined by ELISA. Each value represents the mean ± SEM of five separate experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of SB203580 concentrations. (D) Cells were incubated for 5 h in 3-O- acetyloleanolic acid and SB203580, and Fgf21 mRNA levels were determined by qRT-PCR. Fgf21 mRNA levels were normalized relative to 18S mRNA levels. Each value represents the mean ± SEM of five experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of SB203580 concentrations.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Western Blot, Software, Comparison, Control, Luciferase, Activity Assay, Construct, Cotransfection, Expressing, Plasmid Preparation, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 6. 3-O-acetyloleanolic acid up-regulates FGF21 expression concomitant with the up-regulation of Nr4a1 in C2C12 myotubes. (A) Nr4a1 mRNA expression was up-regulated by 3-O-acetyloleanolic acid in C2C12 myotubes. Cells were treated with 30 µM 3-O-acetyloleanolic acid for 1, 2 and 5 h, harvested, and then processed for real-time qRT-PCR. Each value represents the mean ± SEM of four separate experiments. *P < 0.05, comparison with vehicle control. (B) Cells were incubated for 5 h with 3-O-acetyloleanolic acid and SB203580, and Nr4a1 mRNA levels were determined by qRT-PCR. Nr4a1 mRNA levels were normalized relative to 18S mRNA levels. Each value represents the mean ± SEM of four separate experiments. *P < 0.05, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, com parison of SB203580 concentrations. (C) Cells were transfected with a wild-type FGF21 promoter construct (mFgf21pro1-luc) or NBRE-mutated version (NBRE-mut- mFgf21pro1-luc), and relative luciferase activity was measured. Data are means ± SEM of four separate experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of constructs.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Expressing, Quantitative RT-PCR, Comparison, Control, Incubation, Transfection, Construct, Luciferase, Activity Assay